Activation of the kappa opioid receptor (KOPR) produces many effects including analgesia, dysphoria / aversion, sedation, water diuresis, antipruritic effects and hypothermia. The selective KOPR agonist nalfurafine is used for treatment of uremic pruritis in kidney dialysis patients. Kappa agonists may be useful as analgesics and water diuretics. KOPR antagonists may be useful as antidepressants and anti-anxiety drugs and in alleviating drug craving in addicts. In vitro studies showed that repeated activation of the KOPR (usually epitope-tagged) resulted in reduced responses as well as KOPR internalization and downregulation. However, it has been difficult to examine if the KOPR undergoes similar regulation in vivo and, if so, whether receptor regulation correlates with changes in behavioral responses. It is also challenging to investigate whether KOPR expression and/or trafficking in vivo are altered in brain regions in behavior paradigms (such as stress and chronic drug abuse) that result in KOPR-mediated dysphoria-, depression- and anxiety-like responses, such as after stress exposure or chronic use of drugs of abuse. The difficulty is due to lack of appropriate KOPR antibodies. KOPR antibodies from different labs yielded different results. Here we propose to generate a mouse line expressing the KOPR fused with a fluorescent protein to circumvent the need for specific antibodies. A knockin mouse line expressing the delta opioid receptor fused with the enhanced green fluorescent protein (DOPR-eGFP) has been generated by Kieffer and colleagues and proven to be useful for relating in vivo DOPR trafficking with changes in DOPR-mediated behavior responses. We have generated two mouse KOPR (mKOPR) cDNA constructs, mKOPR-eGFP and mKOPR-tdTomato (mKOPR-tdT). Our preliminary results showed that conjugation with eGFP or tdT did not change the properties of the KOPR when expressed in Neuro2A (N2A) mouse neuroblastoma cells. The specific aims are as follows. (1) Investigate fully if fusion with eGFP or tdT affects expression, ligand binding, signaling and agonist-induced regulation of KOPR in vitro. FLAG-mKOPR will be used for comparison. (2) Generate a knockin mouse line expressing KOPR-eGFP or KOPR-tdT, depending on our in vitro results. Generation of knockin mice will be done in collaboration with the team of Dr. Brigitte Kieffer (Institut de Gntique et de Biologie Molculaire et Cellulaire, Illkirch, France). We choose to collaborate with her because of her track record of success in generating mutant mice, in particular the generation of DOPR-eGFP knockin mice. The targeting strategy will be similar to that used by Kieffer and colleagues. Initial characterization of the knockin mice includes distribution in the brain and spinal cord and ligand binding and signaling of KOPR fusion protein in the brain. The animals will be very useful for correlating KOPR trafficking with changes in KOPR-mediated behaviors and in vivo KOPR internalization may be used as an indicator of KOPR activation. Such a knockin mouse line will be a valuable resource for researchers interested in KOPR trafficking under pharmacological and pathophysiological conditions.